Carica papaya L. sex chromosome review and physical mapping of the serk 2, svp-like and mdar 4 sequences

Physical mapping evidences the chromosome organization and structure. Despite the data about plant cytogenomics, physical mapping has been conducted from single-copy and/or low-copy genes for few species. Carica papaya cytogenomics has been accomplished from BAC-FISH and repeatome sequences. We aimed to map the serk 2, svp-like and mdar 4 sequences in C. papaya. The sequences were amplified and the amplicons sequenced, showing similarity in relation to serk 2, svp-like and mdar 4 genes. Carica papaya diploidy was confirmed and the mitotic chromosomes characterized. The chromosome 1 exhibited the secondary constriction pericentromeric to the centromere of the long arm. So, we concluded that it is the sex chromosomes. serk 2 was mapped in the long arm interstitial portion of the sex chromosomes, and the interphase nuclei showed two fluorescence signals. Considering these results and the sequencing data from the C. papaya sex chromosomes, svp-like and mdar 4 genes were mapped in the interstitial region of the sex chromosome long arm. Both sequences showed only one fluorescence signal in the interphase nuclei. The procedure adopted here can be reproduced for other single-copy and/or low-copy genes, allowing the construction of cytogenetic maps. In addition, we revisited the cytogenomics data about C. papaya sex chromosomes, presenting a revised point of view about the structure and evolution to these chromosomes.

Sex differentiation region in C. papaya genome was genetically mapped to linkage group 1, which is related to the chromosome 1 11,12 .C. papaya biological sex is expressed by a XY chromosome sex differentiation system, from which females are homogametic (XX), and males (XY) and hermaphrodites (XYh) are heterogametic.Y and Yh chromosomes have a specific sexual differentiation region that shows few genetic differences 13 .Malespecific region (MSY) and hermaphrodite-specific region (HSY) are about 9.8 Mbp, while X-specific region (XX) has 6.0 Mbp 14 .MSY and HSY chromosome portions have a larger genome size in relation to the corresponding region on the X chromosome, mainly due to the DNA sequence duplications and insertion of mobile elements (retrotransposons) in this region 13 .HSY possesses more repetitive sequences than X.In addition, 121 pseudoautosomal genes occur between HSY and X chromosomes, 56 specific HSY genes, and 74 specific X genes 14 .
In plants, physical mapping has contributed to the characterization of the sex chromosomes.The mapping of a repetitive DNA sequence, named HSR1, in Humulus lupulus L. showed that this sequence is located in the subtelomeric region of the X and Y chromosome long arm.However, the HSR1 was also mapped in the pericentromeric portion of the chromosome X 18 .In Cannabis sativa L., the repetitive sequence named CS-1 was mapped in the subtelomeric region of the Y chromosome short arm, while both arms of the X chromosome showed this sequence in the subtelomeric region 19 .The mapping of repetitive sequences in Hippophae rhamnoides L. revealed the HRTR 12 repetitive sequence only in the Y chromosome 20 .Repetitive DNA sequences, named RAYS, were only mapped in the Y chromosome of Rumex acetosa L. 21 .Therefore, the sex chromosome characterization in plants has been conducted mainly from the repeatome sequences.In addition to physical mapping, FISH has been accomplished to assists in the early identification of sporophyte sex.For example, a polymorphic molecular marker named NAPF-2 showed fluorescent signals in leaf nuclei of hermaphrodite plants, however, no signal was detected in leaf nuclei of female plants of C. papaya 'Golden' and 'Rubi' 22 .
In addition to these sequences (mainly from the repeatoma), the physical mapping of single-copy and/or low-copy genes is needed to expand the knowledge about the C. papaya genome, contributing to knowing and understanding its organization and evolution.Moreover, these genes also have potential as cytomolecular markers.In this context, some sequences that occur in the sexual differentiation region were explored here, including: somatic embryogenesis receptor kinase gene (serk 2), short vegetative phase gene (svp-like) and monodehydroascorbate reductase gene (mdar 4) 12,23 .
The serk 2 gene, which was sequenced in the X and Yh chromosomes 14 , encodes a protein that belongs to the plasma membrane receptor kinase family.SERK protein has leucine-rich repeats, acting on signal transduction 23 and male sporogenesis 24 .The svp-like gene encodes a transcription factor that regulates the transition from the vegetative to the reproductive phase, activating the classes B and C genes of the floral morphogenesis 25,26 .Gene expression and sequencing approaches showed that the svp-like gene present on the Y chromosome encodes a wild-type protein with all domains.Due to a Copia-like retrotransposon insertion, a mutant allele of this gene occurs on the Yh chromosome, encoding a protein that has only the K-box domain 12,13,27,28 .In addition, svp-like gene was also identified on the chromosome 4 14 .The mdar 4 gene, which was also sequenced in X and Yh chromosomes 14 , encodes an enzyme that has antioxidant activity, eliminating reactive oxygen species and, consequently, increasing tolerance against oxidative stress in some plant species 29,30 .Genetic analyzes have shown that the mdar 4 gene has a wild-type allele on the X chromosome, while the allele of the Y and Yh chromosomes contains a LTR-retroelement sequence 30 .Y and Yh mutant alleles encode a shorter truncated MDAR 4 enzyme in flower tissues 28 .
Considering the previous genomic and cytogenomic data, we hypothesize that C. papaya sex chromosomes differ in several DNA sequences 31 , including svp-like and mdar 4 genes.Based on this, we aimed to map the serk 2, svp-like and mdar 4 sequences in C. papaya mitotic chromosomes.So, we start the mapping of single-copy and/or low-copy genes, differing from the previous C. papaya cytogenomics that were accomplished from rDNA genes and BAC sequences.

Results
The immersion of seeds in GA 3 solution provided a high germination rate (95%) after ~ 5 days at 30 °C.Break seed dormancy was essential to increase the germination rate in a short period, providing a large amount of root meristems to obtain mitotic cells.Prometaphases and metaphases with morphologically preserved chromosomes exhibiting well-defined primary and secondary constrictions were obtained from the cytogenetics procedure.Therefore, we considered adequate the root meristem treatment with amiprophos-methyl/dimethyl sulfoxide, the enzymatic maceration, and the slide preparation accomplishing macerated root dissociation and air drying.The prometaphases and metaphases showed 2n = 2x = 18 chromosomes, confirming the C. papaya diploidy.The mean total chromosome length ranged from 4.32 ± 1.53 µm (chromosome 1) to 2.70 ± 0.42 µm (chromosome 9).Carica papaya karyogram possesses four metacentric (1, 5, 7 and 8) and five submetacentric chromosomes (2, 3, 4, 6 and 9, SI Table 1).Secondary constriction was identified in the pericentromeric region of the chromosome 1 (Fig. 1).
Sequences with ~ 1,100, ~ 250 and ~ 700 bp were amplified from C. papaya genomic DNA and serk 2, mdar 4 and svp-like primers, respectively (SI Fig. 1).The similarity of the amplified serk 2 sequence in relation to C. papaya 'Sunset' and 'SunUp' genomes 14 was 85% for sense strand and 82% for antisense strand in relation to sequenced chromosome 1, and 83% for sense strand and 81% for antisense strand in relation to chromosome Yh.For mdar 4 sequence, the similarity was 95% for sense strand and 94% for antisense strand in relation to the chromosome 1, and 90% for sense strand and 89% for antisense strand in relation to the chromosome Yh.The similarity of the svp-like sequence was 100% for sense strand and 99% for antisense strand in relation to the chromosome 4, and 86% for sense strand and 82% for antisense strand in relation to the chromosome Yh.
We defined the FISH procedure based on different tests and their respective results, considering the visualization of fluorescence signals in nuclei and mitotic chromosomes.We used 4X SSC in the post-hybridization wash solution (stringency).Thus, the FISH conditions were established for the serk 2, svp-like and mdar 4 probes, which showed different sizes in bp.The hybridizations were performed in prometaphases and metaphases to map these sequences, and also in interphase nuclei to confirm the copy number of each sequence.So, clear signals were generated after hybridization of the probes in nuclei and chromosomes.
The serk 2 sequence was mapped in prometaphases/metaphases on the interstitial region of the long arm of chromosome 1 pair, which was identified from the secondary constriction in pericentromeric region.This chromosome has been appointed as the sex chromosome of C. papaya.In addition, two fluorescence signals were detected in interphase nuclei, confirming that there is one copy of the serk 2 in the C. papaya genome.As well as serk 2, svp-like and mdar 4 sequences were mapped in prometaphases/metaphases in the interstitial region of the long arm of chromosome 1.However, only one chromosome 1 exhibited these sequences (Figs. 1 and 2).Corroborating this, we found one svp-like and mdar 4 fluorescence in interphase nuclei.Thus, our results suggest differences between the chromosomes of the homologous pair 1, also designated as X and Y/Yh.
Based on the karyograms (Fig. 1), we structured an ideogram of the chromosomes X and Y/Yh, representing the loci marked by the serk 2, mdar 4 and svp-like, as well as the centromere and the secondary constriction (Fig. 3).The results obtained in this study increase the cytogenomic data of C. papaya through the physical mapping of the serk 2, mdar 4 and svp-like.In addition, the ideogram includes the cytogenomic data of the physical location of heterochromatic portions 32 , the 5S rDNA gene 15 and the distribution of retroelements 27 .   .The dark blue circle in the pericentromeric region of the long arm of the Y chromosome represents the 5S rDNA 15 .The green circles show the distribution of retroelements along the sex chromosomes 27

Discussion
We mapped the serk 2, mdar 4 and svp-like, providing the first physical mapping of these sequences in C. papaya mitotic chromosomes.Accomplishing the same FISH procedure, we mapped amplicons from 251 to 1166 bp, resulting in a physical mapping in mitotic chromosomes from sequences of single-copy or low-copy in C. papaya.Our procedure can be reproduced allowing the physical mapping of genes with single-copy or low-copy.In addition, our results represent the start point for further physical mapping, which are relevant to increase knowledge about the C. papaya genome.We integrated the physical map with the in silico data, contributing to knowing and understanding of the sexual chromosomes X and Y/Yh of the C. papaya.In addition, the svp-like can be further used as cytomolecular markers to previously screen the sex of C. papaya plants.
C. papaya is a model species for studies about sex chromosome evolution and sex differentiation in plants, mainly due to incipient evolution of its sex chromosomes X and Y/Yh, and the recombination suppression in the non-homologue portion between them 1,5 .In addition to the svp-like and mdar 4 sequences, a relatively high density of retroelements was identified along the C. papaya sex chromosomes 27 .Furthermore, at least four knobs (heterochromatic portions) and highly methylated sequences were identified only in the sexual region of the Y/Yh chromosome and not in the non-homologous portion of the X chromosome 32 .These cytogenomic characteristics may influence gene expression and sequence divergence.
serk 2 physical mapping revealed its locus in the interstitial region of the chromosome 1 long arm.The sequencing of the X and Y/Yh sex chromosome dedifferentiation portions showed that the serk 2 gene is present in this region 12,14 .The presence of two signals in the nuclei and in the metaphases confirms that the serk 2 is a single-copy gene, and that it possibly occurs near or in the pseudoautosomal portion of the X and Y/Yh chromosomes.Although the sex determination region has specific genes or sequences of each sex chromosome (74 genes in X, and 56 genes in Yh), at least 121 genes have been predicted in the sex region of the X chromosome and in its Yh counterpart 13,14 .mdar 4 sequence, which was amplified from C. papaya genomic DNA, locus was also mapped in the chromosome 1 interstitial region, and only one hybridization signal of this sequence was detected in the nuclei and chromosomes.Genomic analyses showed that the divergence between the mdar 4 gene in sex chromosomes is due to the insertion of retroelements inside of this gene in Y (male) and Yh (hermaphrodite) chromosomes, which does not occur in X chromosome 27 .Since the mdar 4 gene plays a role in eliminating reactive oxygen species, maintaining cell viability, the absence of this gene on the X chromosome can be lethal 30 .The alignment of a region of the X and Yh C. papaya chromosomes revealed deletions on the Yh.For example, the asymmetric leaves 2 gene appears to have been lost, since it occurs only on the X chromosome 10 .Gene loss has been reported in Silene latifolia Poir.Y chromosome, which lossed ~ 14.5 of genes 33 .Thus, further studies in C. papaya, including cytogenomics, need to be conducted to show possible gene loss between the X and Y/Yh chromosomes.
svp-like was mapped in the interstitial region of the 1 chromosome long arm.The transcriptome indicated that svp-like gene occurs only on the sex differentiation region of the Y (male) and Yh (hermaphrodite) chromosome 27 .svp-like gene was also identified by sequencing in the chromosome 4 14 .The sequencing showed that our svp-like sequence possesses similarity in relation to svp-like gene of the Yh chromosome.Therefore, our data suggest that the svp-like sequence was mapped in the Y/Yh chromosome.
The mapping of the svp-like and mdar 4 sequences on chromosome 1, which shows the secondary constriction and the higher total length.So, this result corroborates with sex differentiation region in C. papaya, which is in a pericentromeric region that expands to the long arm of this chromosome 33 .The expansion of the suppressed recombination region occurs from the selection of sexual antagonistic genes and/or chromosomal rearrangements [33][34][35] .As a consequence of the recombination suppression in the sex differentiation region, the Y/Yh chromosome undergoes genetic degeneration, presenting low expression or loss of genes.Suppressed recombination in sex differentiation region can promote divergence between the sequences present in this chromosomal portion and gradually change its copy number.
The identification of cytogenetic differences and the characterization of sex chromosomes in plants have been conducted mainly through physical mapping of repetitive sequences.In H. lupulus, the satellite DNA HSR1 was mapped in the subtelomeric and pericentromeric region of the X chromosome, while the Y chromosome presented this sequence only in the subtelomeric region 18 .The mapping of the repetitive sequence CS-1 differentiated the sex chromosomes of C. sativa, due to this sequence occurs only in the subtelomeric region of the Y chromosome short arm and in the subtelomeric region of both arms of the X chromosome 18 .In H. rhamnoides and R. acetosa, the satellite DNA sequences HRTR 12 and RAYS, respectively, were mapped only on the Y chromosome 20,21 .
In addition to the sex chromosomes characterization, molecular cytogenetics have been accomplished to determine the plant sex before the reproductive period.Based on our results and previous sequencing data, the svp-like sequence can be explored as cytomolecular markers to early determine the sex of C. papaya plants.In C. papaya, molecular markers of the Sequence Characterized Amplified Region type (SCAR) were used to differentiate hermaphrodite individuals from females, through the FISH application in nuclei isolated from the leaves.Using the molecular marker NAPF-2, only the nuclei isolated from the hermaphrodite plants showed a strong fluorescence signal, which was not observed in the leaf nuclei of female plants 22 .Thus, FISH is also useful to early determine and screen the sex, reducing the growing costs of the sex of noncommercial interest.
For the first time, single-copy sequences were mapped in C. papaya sex chromosomes.We mapped serk 2, svplike and mdar 4 sequences, which are single-copy or low-copy sequences, in mitotic chromosomes of C. papaya.Therefore, our data represents the start point to map other specific sex chromosome genes, increasing cytogenomics analyzes and the understanding of the evolution and structure of the sex chromosomes in C. papaya.

FISH
Carica papaya total genomic DNA was extracted from young leaves using GenElute™ Plant Genomic DNA Miniprep Kit (Sigma ® ), following the manufacturer's instructions.DNA concentration and purity were determined by a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific ® ), and its integrity was evaluated by electrophoresis in a 1.5% agarose gel.The primers of the serk 2 and mdar 4 were designed based on the sequences deposited at the National Center for Biotechnology Information (NCBI): serk 2-LOC110813265 and mdar 4-LOC110807062.For the svp-like, the primers were obtained based on the bibliography 34 .So, we designed the primers: F 5′-CTC TCA CTG CAC GCC TAA C-3′ and R 5′-TCG CCT TCA AAT CCT GAA ACT-3' for serk 2 providing a 1,166 bp amplification product; F 5′-ACT TGT TGC CTC AGT TTC TCA TTC TCTTC-3′ and R 5′-GAG ATC AGT GAT CTT CAA AGG AAG GTC-3′ for svp-like resulting a 251 bp amplification product; and F 5′-TAT TCC GAC CCC AGT CTC CA-3' and R 5'-TCC TAC CGC GCC AAA CAA AT-3′ for mdar 4 providing a 731 bp amplification product.The primers were validated in silico using the OligoAnalyzer™ tool (https:// www.idtdna.com/ pages/ tools/ oligo analy zer), and the formation of homodimers, heterodimers and hairpin structures were verified.Specificity analysis was performed using the Basic Local Alignment Search Tool (BLAST, https:// blast.ncbi.nlm.nih.gov/ Blast.cgi).
Slides with prometaphases and metaphases exhibiting chromosomes with well-defined centromeres were selected from phase contrast microscope Olympus BX41.The selected slides were treated in 1X phosphatebuffered saline (PBS) for 5 min, fixed in 4% formalin solution for 10 min, and washed with 1X PBS for 5 min.Subsequently, the slides were dehydrated in an ice-cold alcoholic series (70%, 85% and 100%) for 3 min each.The hybridization mix consisted of 200 ng of each probe, 50% formamide and 2X saline sodium citrate (SSC).Chromosomes were denatured in a water bath at 70 °C for 3 min in 70% formamide/2X SSC.After denaturation, the slides were dehydrated in an ice-cold alcoholic series (70%, 85% and 100%) for 3 min each.The hybridization mix was denatured in a PTC-200 Peltier Thermal Cycler (MJ Research, Inc) at 85 °C for 5 min and immediately placed on ice for at least 5 min.The hybridization mix was placed on the slides, covered with HybriSlip™ plastic coverslips (Sigma ® ) and sealed with Rubber Cement glue (Elmer's ® ).Hybridization was carried out in the ThermoBrite™ equipment (StatSpin ® ) at 37 °C for 24 h.Subsequently, the slides were submitted to a stringency wash performed at 40 °C in 4X SSC solution for 5 min.Slides were counterstained with 10% glycerol/ PBS + 4′,6-diamidino-2-phenylindole (DAPI), covered with a glass coverslip and sealed with nail polish [39][40][41][42] .Nuclei, prometaphases and metaphases were captured with a 12-bit CCD digital camera (Olympus ® ) attached to an Olympus BX-60 photomicroscope equipped with epifluorescence and 100 × PlanApo immersion objective (NA = 1.4).Captured images were processed using Image-Pro Plus 6.1 software (Media Cybernetics, Inc).

Figure 1 .
Figure 1.Physical mapping of the serk 2, mdar 4 and svp-like sequences in mitotic chromosomes of C. papaya.Detail for the markings of the serk 2, mdar 4 and svp-like in the interstitial region of the chromosome 1 long arm.The karyograms show 2n = 2x = 18 chromosomes, aligned by the centromere.The secondary constriction present in the pericentromeric region of chromosome 1 is observed.Bars: 10 μm.

Figure 2 .
Figure 2. Copy number of the serk 2, mdar 4 and svp-like sequences in interphase nuclei of C. papaya.Two signals were detected for serk 2, and one for mdar 4 and svp-like.

Figure 3 .
Figure 3. Ideograms of the X and Yh/Y chromosomes of C. papaya.Chromosomes 1 exhibiting the fluorescence signal for serk 2, mdar 4 and svp-like sequences.The orange bar represents the position of the centromere.The light blue circle shows the position of the secondary constriction.The pink and purple circles on the long arm of the X chromosome show the loci of the serk 2 and mdar 4 sequences, respectively.The pink, purple and red circles on the long arm of the Yh/Y chromosome shows the loci of the serk 2, mdar 4 and svp-like genes.The yellow circles represent the knobs (heterochromatic portions)39 .The dark blue circle in the pericentromeric region of the long arm of the Y chromosome represents the 5S rDNA15 .The green circles show the distribution of retroelements along the sex chromosomes27 .